Time-resolved optical spectroscopy with nanosecond lasers and molecular dynamics calculations are being employed to investigate ligand rebinding and conformational changes in hemoglobin subsequent to photodissociation of the carbon monoxide complex. These methods have been used to determine kinetic parameters for geminate rebinding in hemoglobin in both the R and the T quaternary structures, to measure the rate of the R to T structural change, and to measure the kinetics of the tertiary conformational changes resulting from ligand dissociation in both quaternary structures. Comparison of activation and equilibrium parameters shows that the transition state for the quaternary transition is much more R-like than T-like, explaining the linear free energy relation between quaternary rates and equilibria. The R-like transition state may be explained by a reaction path which maximizes the buried surface area. In a related project it has been shown that single crystals of hemoglobin in the T quaternary structure bind oxygen non-cooperatively with no Bohr ii effect, a result that has several important implications for the molecular mechanism of cooperativity. Studies are being carried out to provide a quantitative description of the gelation of hemoglobin S that can be used for understanding the pathophysiology of sickle cell disease and the development of therapeutic agents. Measurements of gelation delay times and domain density indicate that the rate of domain formation is comparable to the rate of homogeneous nucleation predicted by the double nucleation model for polymer formation. Optical micrographs of cells deoxygenated at various rates indicate that the morphology of cells is controlled by the density of polymer domains, and, therefore, the rate of homogeneous nucleation. Thus the delay time of gelation not only determines whether intracellular polymerization will occur in vivo, but it also determines the shape of the resulting sickled cell. This information is critical to the design of an automated "sickling assay" that will make it possible to examine a large number of potentially therapeutic agents, to compare intracellular gelation and clinical severity, and to follow changes in intracellular gelation in patients on various therapeutic protocols.